UC Irvine

Two critical steps in mRNA processing are 3’ processing and splicing, both of which diversify the human genome and create mRNA isoforms with unique regulatory properties including mRNA stability, translation efficiency or intracellular location or even create distinct proteins. In addition to being essential steps in mRNA processing, polyadenylation and splicing are highly alternatively regulated, with approximately 70% and 95% of genes producing alternative isoforms respectively. Regulation of these isoforms have important biological consequences as mis-regulation is associated with many human diseases including cancer and neurological disorders (Tian and Manley 2017; S. Chan, Choi, and Shi 2011; Q. Pan et al. 2008; Lukong et al. 2008; Y. Zhang et al. 2021). While the core regulatory machineries for 3’ processing and splicing have been identified, there is still limited ability to predict how alternative regulation will occur, making this a critical field of study. One area of particular interest for the study of alternative RNA processing is the role of RNA binding proteins (RBPs). To better understand how RNA binding proteins regulate alternative 3’ processing and splicing, I carried out three projects. First, to investigate the roles of RNA binding proteins in 3’ processing regulation, a dual luciferase reporter system was utilized for a large-scale screen of RBPs for polyadenylation site regulation. In addition to validating several known regulators of APA, we identify several novel inhibitors of polyA site selection including hnRNP A0, hnRNP G, and Musashi1. Strikingly, we also demonstrate that the SR family of proteins are polyA site position-independent repressors of polyadenylation sites, indicating that their role in polyadenylation may be unique from their role in splicing regulation where they act as position dependent activators. This screen may also be used to identify other families of RBPs with the capability to regulate polyadenylation and make predictions about other new regulators. Next, genome-wide sequencing approaches were used to characterize the role of the core polyadenylation complex cleavage factor I (CFIm) in APA regulation. In addition to its known role in enhancing distal polyA sites, we demonstrate that CFIm promotes intronic polyadenylation, most notably within one member of each of the other core polyadenylation machinery components. We also propose a model by which CFIm regulates both 3’ UTR APA and intronic polyadenylation to modulate global protein production and therefore link 3’ processing with cell fate determination. Finally, we interrogate the role of CFIm in splicing regulation through a combination of genome-wide sequencing and biochemical analyses. We demonstrate that CFIm is a general alternative splicing regulator that binds 3’ splice site and interacts with U2AF through RS-RS domain interactions. In addition, CFIm promotes U2AF-RNA interaction at the 3’ splice sites of CFIm activated cassette exons. Our data supports a revised model for 3’ splice site selection by U2AF: CFIm and other RNA binding proteins compete for interaction with U2AF and each regulate the alternative splicing of a specific subset of cassette exons.